The publication and other material used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The polymerase chain reaction (PCR) (Saiki et al., 1985 Science 230, 1350-1354) is a nucleic acid amplification technique that has become the most important nucleic acid diagnostic tool. It enables extremely sensitive detection of specific nucleic acid sequences in various sample matrices. To find out whether or not a sample contains e.g. a specific pathogen, the sample can be analyzed for the presence of a nucleic acid specific to the pathogen by PCR. If, using oligonucleotide primers specific for the nucleic acid of the pathogen of interest, a PCR product can be amplified starting from nucleic acids extracted from the sample, the sample is likely to contain the pathogen of interest. Since PCR can, at least in theory, amplify even one target DNA molecule up to a detectable level, it allows extremely sensitive detection of pathogens, mutations, cancer cells and other targets that can be identified by specific nucleic acid sequences. In order for the assays to function, all PCR tests require a method for reliable and accurate detection and identification of the PCR product. The first diagnostic tests based on PCR were quite cumbersome and not amenable to large scale screening methods, since the PCR detection methods were not very straightforward. Several post PCR steps, such as restriction enzyme analysis, agarose gel electrophoresis or heterogeneous hybridization assays were needed to confirm the identity of the PCR product. These methods require that the reaction vessels in which PCR is performed are opened after amplification, which constitutes a serious risk of contamination and consequent false positive PCR results. To overcome the problems associated with manipulation of PCR products, fluorescent techniques and assay formats have been developed that greatly simplify the protocols used for the detection of specific nucleic acid sequences. These methods, exemplified e.g. in U.S. Pat. Nos. 5,994,056, 5,804,375, EP0543942, EP0232967, US2003143591, US2003022177, US2004029119, EP0912760 and by Sanchez J. et al. in “Two-temperature LATE-PCR endpoint genotyping”, published in BMC Biotechnology, vol. 6, December 2006, involve the detection of a specific PCR product in a homogeneous solution without the need to open the amplification tubes after PCR. The results can be read in real time as the PCR product is accumulated or at the end of the thermal cycling protocol directly from the closed amplification wells.
The principle of real-time PCR is described e.g. in U.S. Pat. No. 5,994,056. In real-time PCR, fluorescence generated by an intercalating dye or by a homogeneous probe-based detection system is measured more than once during PCR amplification. Typically, the first measurement or measurements are performed in the beginning or even prior to target amplification to determine the baseline signal of the reaction. To determine whether or not the target sequence is amplified, fluorescent signal intensities obtained later during amplification or after amplification has been completed are compared to the baseline and, if a significant change—an increase or a decrease, depending on the detection method that is used—is detected, the reaction is considered to be positive, i.e. to contain the sequence of interest. On the other hand, if there is no significant change in the intensity of the signal recorded from the reaction, the reaction is considered negative i.e. not to contain the sequence of interest. In essence, real-time PCR is thus based on detecting a change in signal intensity, said change being caused by the appearance of the specific PCR product. The clear benefit of the method is that a reaction-specific baseline level can be determined to which all subsequent signal intensities can be compared. This makes the technique very sensitive. However, in the measurement of a change in fluorescence lies a problem: changes in fluorescence intensity can be caused by artifacts that are not related to the specific amplification reaction. For example, a leaking reaction vessel lid or appearance of bubbles in the reaction solution during thermal cycling can cause significant changes in the measured signals without any relation to the amplification process. Also, it may be difficult to detect the specific change in signal in the presence of a lot of background fluorescence. Such unspecific effects on fluorescence intensity can, in the worst case, result in false results.
Instead of using the real-time measurement technique, one can also determine whether or not a specific nucleic acid sequence has been amplified by performing a measurement after completion of the amplification protocol, provided that the reaction mixture includes an intercalating dye or a probe system capable of reporting the presence of a specific target sequence. In such end-point assays it is common practice to analyze negative control reactions in parallel with the actual samples. The negative control reactions are usually prepared by adding water instead of a template nucleic acid to an amplification mixture—therefore, no amplification of the target nucleic acid takes place in the negative control reactions. Thus, the negative control reactions are used to determine the baseline signal that is characteristic for the batch of analytical results—it therefore plays the role in end-point assays that the initial baseline measurements have in the real-time technique. Negative control reactions need to be included in each analytical run since the fluorescence background emitted by individually prepared reaction mixtures varies to some extent and, even more importantly, the absolute signal levels recorded by individual fluorescence measurement instruments varies. Therefore, it is not possible to determine a general background level that would be applicable in all instruments at all times. To determine whether or not a sample contains the sequence of interest, the signal emitted by the sample reaction is compared to the negative controls. If a significant difference is detected between the signals emitted by the sample reactions and the negative control reactions, the sample is considered positive. On the other hand, if the sample reaction gives a signal intensity that is essentially the same as the signal given by the negative control reactions, the sample is considered negative. Therefore, this method is also based on detecting a change in fluorescence intensity—the main difference between this technique and the real-time technique is that while in the real-time method the baseline is determined for each reaction individually, in the end-point method a common baseline is determined for all simultaneously analyzed samples using negative control reactions that are run in parallel with the sample reactions. Just like in the real-time method, false results can be caused by unspecific sources of fluorescence change that may take place in the negative control reactions or in the sample reactions. Furthermore, results can be distorted by differences in the background signal emitted by individual samples: if, for example, a particular sample contains a colored substance that affects the fluorescence emitted by the fluorophores utilized in the detection method, false results can be obtained. Another important source of errors is that the method is very sensitive to the exact reaction volume—even slight changes in reaction volume can distort the results if individual control and sample reactions contain slightly different amounts of fluorescent label to begin with.
One solution to unspecific changes in fluorescence intensity caused by differences in reaction volume has been described in U.S. Pat. No. 5,928,907. In the method described in U.S. Pat. No. 5,928,907 each reaction contains—in addition to a first fluorescent indicator, the signal intensity of which is related to the amount of PCR product present in the reaction—a second fluorescent indicator, which is a label molecule the signal intensity of which is essentially independent of the amount of PCR product present in the reaction. Instead, the signal intensity of the second fluorescent indicator depends on the reaction volume in a similar manner as the first fluorescent indicator. Therefore, by recording at each measurement the signals of both fluorescent indicators, it is possible to eliminate the unspecific effects on signal intensities caused by differences in reaction volume by correcting the signals of the first fluorescent indicator by calculating the relationship between the signals given by the first and second fluorescent indicators. While this method has found wide acceptance and applications in the art, it has the intrinsic problem that the second fluorescent indicator as such increases the total fluorescence background of the reaction and reduces the possibilities for multiplexing. In this context, multiplexing means the art of combining the amplification and detection reactions of several different target nucleic acids in one PCR reaction. If a second fluorescent indicator according to U.S. Pat. No. 5,928,907 is used, the spectral area of the second fluorescent indicator is reserved, leaving less room for other fluorescent indicators allowing the detection of other target sequences in the same reaction. That is, if one has access to nine different spectrally resolvable labels, one can maximally only amplify and detect eight different targets simultaneously, if one of the labels has to be used as a second fluorescent indicator. It would be desirable to be able to combine as many targets as possible in one reaction—therefore, it would be advantageous if the second fluorescent indicator was not needed.
Therefore, due to the problems associated with the existing techniques, there is a need for a method that would allow the detection of a PCR product without the need to resort to real-time measurement, negative control reactions or a second fluorescent indicator dye. These problems are solved with the methods of the present invention.